Dataset Information

Induction of Shiga Toxin-Encoding Prophage by Abiotic Environmental Stress in Food.

ABSTRACT: The prophage-encoded Shiga toxin is a major virulence factor in Stx-producing Escherichia coli (STEC). Toxin production and phage production are linked and occur after induction of the RecA-dependent SOS response. However, food-related stress and Stx-prophage induction have not been studied at the single-cell level. This study investigated the effects of abiotic environmental stress on stx expression by single-cell quantification of gene expression in STEC O104:H4 ?stx2::gfp::amp^r In addition, the effect of stress on production of phage particles was determined. The lethality of stressors, including heat, HCl, lactic acid, hydrogen peroxide, and high hydrostatic pressure, was selected to reduce cell counts by 1 to 2 log CFU/ml. The integrity of the bacterial membrane after exposure to stress was measured by propidium iodide (PI). The fluorescent signals of green fluorescent protein (GFP) and PI were quantified by flow cytometry. The mechanism of prophage induction by stress was evaluated by relative gene expression of recA and cell morphology. Acid (pH < 3.5) and H₂O₂ (2.5 mM) induced the expression of stx₂ in about 18% and 3% of the population, respectively. The mechanism of prophage induction by acid differs from that of induction by H₂O₂ H₂O₂ induction but not acid induction corresponded to production of infectious phage particles, upregulation of recA, and cell filamentation. Pressure (200 MPa) or heat did not induce the Stx2-encoding prophage (Stx2-prophage). Overall, the quantification method developed in this study allowed investigation of prophage induction and physiological properties at the single-cell level. H₂O₂ and acids mediate different pathways to induce Stx2-prophage.IMPORTANCE Induction of the Stx-prophage in STEC results in production of phage particles and Stx and thus relates to virulence as well as the transduction of virulence genes. This study developed a method for a detection of the induction of Stx-prophages at the single-cell level; membrane permeability and an indication of SOS response to environmental stress were additionally assessed. H₂O₂ and mitomycin C induced expression of the prophage and activated a SOS response. In contrast, HCl and lactic acid induced the Stx-prophage but not the SOS response. The lifestyle of STEC exposes the organism to intestinal and extraintestinal environments that impose oxidative and acid stress. A more thorough understanding of the influence of food processing-related stressors on Stx-prophage expression thus facilitates control of STEC in food systems by minimizing prophage induction during food production and storage.

SUBMITTER: Fang Y

PROVIDER: S-EPMC5601330 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Induction of Shiga Toxin-Encoding Prophage by Abiotic Environmental Stress in Food.

Fang Yuan Y Mercer Ryan G RG McMullen Lynn M LM Gänzle Michael G MG

Applied and environmental microbiology 20170915 19

The prophage-encoded Shiga toxin is a major virulence factor in Stx-producing Escherichia coli (STEC). Toxin production and phage production are linked and occur after induction of the RecA-dependent SOS response. However, food-related stress and Stx-prophage induction have not been studied at the single-cell level. This study investigated the effects of abiotic environmental stress on stx expression by single-cell quantification of gene expression in STEC O104:H4 Δstx2:: ...[more]

PMID: 28778890

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Project description:Shiga toxin (Stx), encoded by stx genes located in prophage sequences, is the major agent responsible for the pathogenicity of Shiga toxin-producing Escherichia coli (STEC) and is closely associated with the development of hemolytic uremic syndrome (HUS). Although numerous Stx prophage sequences have been reported as part of STEC bacterial genomes, the information about the genomic characterization of Stx-converting bacteriophages induced from STEC strains is relatively scarce. The objectives of this study were to genomically characterize two Stx-converting phages induced from environmental STEC strains and to evaluate their correlations with published Stx-converting phages and STEC strains of different origins. The Stx1-converting phage Lys8385Vzw and the Stx2-converting phage Lys19259Vzw were induced from E. coli O103:H11 (RM8385) and E. coli O157:H7 (RM19259), respectively. Whole-genome sequencing of these phages was conducted on a MiSeq sequencer for genomic characterization. Phylogenetic analysis and comparative genomics were performed to determine the correlations between these two Stx-converting phages, 13 reference Stx-converting phages, and 10 reference STEC genomes carrying closely related Stx prophages. Both Stx-converting phages Lys8385Vzw and Lys19259Vzw had double-stranded DNA, with genome sizes of 50,953 and 61,072 bp, respectively. Approximately 40% of the annotated coding DNA sequences with the predicted functions were likely associated with the fitness for both phages and their bacterial hosts. The whole-genome-based phylogenetic analysis of these two Stx-converting phages and 13 reference Stx-converting phages revealed that the 15 Stx-converting phages were divided into three distinct clusters, and those from E. coli O157:H7, in particular, were distributed in each cluster, demonstrating the high genomic diversity of these Stx-converting phages. The genomes of Stx-converting phage Lys8385Vzw and Lys19259Vzw shared a high-nucleotide similarity with the prophage sequences of the selected STEC isolates from the clinical and environmental origin. The findings demonstrate the genomic diversity of Stx-converting phages induced from different STEC strains and provide valuable insights into the dissemination of stx genes among E. coli population via the lysogenization of Stx-converting phages.

Dataset Information

Induction of Shiga Toxin-Encoding Prophage by Abiotic Environmental Stress in Food.

Publications

Induction of Shiga Toxin-Encoding Prophage by Abiotic Environmental Stress in Food.

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